Etsuko Shibata

CRL4Cdt2 Regulates Cell Proliferation and Histone Gene Expression by Targeting PR-Set7/Set8 for Degradation

PR-Set7/Set8 is a cell-cycle-regulated enzyme that monomethylates lysine 20 of histone H4 (H4K20). Set8 and monomethylated H4K20 are virtually undetectable during G1 and S phases of the cell cycle but increase in late S and in G2. We identify CRL4(Cdt2) as the principal E3 ubiquitin ligase responsible for Set8 proteolytic degradation in the S phase of the cell cycle, which requires Set8-PCNA interaction. Inactivation of the CRL4-Cdt2-PCNA-Set8 degradation axis results in (1) DNA damage and the induction of tumor suppressor p53 and p53-transactivated proapoptotic genes, (2) delayed progression through G2 phase of the cell cycle due to activation of the G2/M checkpoint, (3) specific repression of histone gene transcription and depletion of the histone proteins, and (4) repression of E2F1-dependent gene transcription. These results demonstrate a central role of CRL4(Cdt2)-dependent cell-cycle regulation of Set8 for the maintenance of a stable epigenetic state essential for cell viability.

Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage

ABSTRACT The role of chromatin-remodeling factors in transcription is well established, but the link between chromatin-remodeling complexes and DNA repair remains unexplored. Human Rvb1 and Rvb2 are highly conserved AAA+ ATP binding proteins that are part of various chromatin-remodeling complexes, such as Ino80, SNF2-related CBP activator protein (SRCAP), and Tip60/NuA4 complexes, but their molecular function is unclear. The depletion of Rvb1 increases the amount and persistence of phosphorylation on chromatin-associated H2AX after the exposure of cells to UV irradiation or to mitomycin C, cisplatin, camptothecin, or etoposide, without increasing the amount of DNA damage. Tip60 depletion, but not Ino80 or SRCAP depletion, mimics the effect of Rvb1 depletion on H2AX phosphorylation. Rvb1 is required for the histone acetyltransferase (HAT) activity of the Tip60 complex, and histone H4 acetylation is required prior to the dephosphorylation of phospho-H2AX. Thus, Rvb1 is critical for the dephosphorylation of phospho-H2AX due to the role of Rvb1 in maintaining the HAT activity of Tip60/NuA4, implicating the Rvb1-Tip60 complex in the chromatin-remodeling response of cells after DNA damage.

Deubiquitination of Tip60 by USP7 Determines the Activity of the p53-Dependent Apoptotic Pathway

ABSTRACT Tip60 is an essential acetyltransferase required for acetylation of nucleosomal histones and other nonhistone proteins. Tip60 acetylates the p53 tumor suppressor at lysine 120 (K120), a modification essential for p53-dependent induction of PUMA and apoptosis. It is known that Tip60 is turned over in cells by the ubiquitin-proteasome system. However, the deubiquitinase activity for stabilizing Tip60 is unknown. Here we show that USP7 interacts with and deubiquitinates Tip60 both in vitro and in vivo. USP7 deubiquitinase activity is required for the stabilization of Tip60 in order to operate an effective p53-dependent apoptotic pathway in response to genotoxic stress. Inhibiting USP7 with the small-molecule inhibitor P22077 attenuates the p53-dependent apoptotic pathway by destabilizing Tip60. P22077, however, is still cytotoxic, and this is partly due to destabilization of Tip60.

Overcoming Platinum Resistance in Preclinical Models of Ovarian Cancer Using the Neddylation Inhibitor MLN4924

The nearly ubiquitous development of chemoresistant disease remains a major obstacle against improving outcomes for patients with ovarian cancer. In this investigation, we evaluated the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, in ovarian cancer cells. Efficacy of MLN4924 both alone and in combination with platinum was assessed. Overall, single-agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. However, the combination of MLN4924 with cisplatin or carboplatin produced synergistic effects in SKOV3 and ES2 cells, as well as in primary ovarian cancer cell lines established from high-grade serous, clear cell, and serous borderline ovarian tumors. The efficacy of cisplatin plus MLN4924 was also evident in several in vitro models of platinum-resistant ovarian cancer. Mechanistically, the combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, abrogation of FANCD2 monoubiquitination, or CHK1 phosphorylation. An siRNA screen was used to investigate the contribution of each member of the cullin RING ligase (CRL) family of E3 ubiquitin ligases, the best-characterized downstream mediators of MLN4924s biologic effects. Cisplatin-induced cytotoxicity was augmented by depletion of CUL3, and antagonized by siCUL1 in both ES2 and SKOV3 ovarian cancer cells. This investigation identifies inhibition of neddylation as a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. Mol Cancer Ther; 12(10); 1958–67. ©2013 AACR.

The MCM8-MCM9 Complex Promotes RAD51 Recruitment at DNA Damage Sites To Facilitate Homologous Recombination

ABSTRACT The minichromosome maintenance protein homologs MCM8 and MCM9 have previously been implicated in DNA replication elongation and prereplication complex (pre-RC) formation, respectively. We found that MCM8 and MCM9 physically associate with each other and that MCM8 is required for the stability of MCM9 protein in mammalian cells. Depletion of MCM8 or MCM9 in human cancer cells or the loss of function MCM9 mutation in mouse embryo fibroblasts sensitizes cells to the DNA interstrand cross-linking (ICL) agent cisplatin. Consistent with a role in the repair of ICLs by homologous recombination (HR), knockdown of MCM8 or MCM9 significantly reduces HR repair efficiency. Chromatin immunoprecipitation analysis using human DR-GFP cells or Xenopus egg extract demonstrated that MCM8 and MCM9 proteins are rapidly recruited to DNA damage sites and promote RAD51 recruitment. Thus, these two metazoan-specific MCM homologs are new components of HR and may represent novel targets for treating cancer in combination with DNA cross-linking agents.

CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration.

The Cul4-Cdt2 (CRL4(Cdt2)) E3 ubiquitin ligase is a master regulator of cell-cycle progression and genome stability. Despite its central role in the degradation of many cell-cycle regulators, e.g., Cdt1, p21, and Pr-Set7/Set8, little is known about the regulation of its activity. We report that Cdt2 is autoubiquitylated by the CRL4A E3 ubiquitin ligase. Cdt2 is additionally polyubiquitylated and degraded by Cul1-FBXO11 (CRL1(FBXO11)). CRL1(FBXO11)-mediated degradation of Cdt2 stabilizes p21 and Set8, and this is important during the response to TGF-β, with the Set8 induction being important for turning off the activation of Smad2. The migration of epithelial cells is also stimulated by CRL1(FBXO11)-mediated downregulation of Cdt2 and the consequent stabilization of Set8. This is an interesting example of cross-regulation between specific Cullin 4 and Cullin 1 E3 ubiquitin ligases and highlights the role of ubiquitylation in regulating cellular responses to TGF-β and the migration of epithelial cells.

Selective Ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G Ubiquitin-Conjugating Enzymes via the CRL4Cdt2 Ubiquitin Ligase Complex

ABSTRACT CRL4Cdt2 is a cullin-based E3 ubiquitin ligase that promotes the ubiquitin-dependent proteolysis of various substrates implicated in the control of cell cycle and various DNA metabolic processes such as DNA replication and repair. Substrates for CRL4Cdt2 E3 ubiquitin ligase include the replication licensing factor Cdt1 and the cyclin-dependent kinase (Cdk) inhibitor p21. Inhibition of this E3 ligase leads to serious abnormalities of the cell cycle and cell death. The ubiquitin-conjugating enzyme (UBC) involved in this important pathway, however, remains unknown. By a proteomic analysis of Cdt2-associated proteins and an RNA interference-based screening approach, we show that CRL4Cdt2 utilizes two different UBCs to target different substrates. UBCH8, a member of the UBE2E family of UBCs, ubiquitylates and promotes the degradation of p21, both during the normal cell cycle and in UV-irradiated cells. Importantly, depletion of UBCH8 by small interfering RNA (siRNA) increases p21 protein level, delays entry into S phase of the cell cycle, and suppresses the DNA damage response after UV irradiation. On the other hand, members of the UBE2G family of UBCs (UBE2G1 and UBE2G2) cooperate with CRL4Cdt2 to polyubiquitylate and degrade Cdt1 postradiation, an activity that is critical for preventing origin licensing in DNA-damaged cells. Finally, we show that UBCH8, but not UBE2G1 or UBE2G2, is required for CRL4Cdt2-mediated ubiquitylation and degradation of the histone H4 lysine 20 monomethyltransferase Set8, a previously identified CRL4Cdt2 substrate, as well as for CRL4Cdt2-dependent monoubiquitylation of PCNA in unstressed cells. These findings identify the UBCs required for the activity of CRL4Cdt2 on multiple substrates and demonstrate that different UBCs are involved in the selective ubiquitylation of different substrates by the same E3 complex.

CRL4Cdt2 E3 Ubiquitin Ligase and Proliferating Cell Nuclear Antigen (PCNA) Cooperate to Degrade Thymine DNA Glycosylase in S Phase

Background: TDG is degraded in S phase via the proteasomal pathway. Results: CRL4Cdt2 ubiquitinates and targets TDG associated with PCNA for degradation. Conclusion: CRL4Cdt2-dependent degradation of TDG is important for preventing toxicity from excess TDG. Significance: This is the first report demonstrating that TDG is regulated by CRL4Cdt2. Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4Cdt2 E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4Cdt2, specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4Cdt2 E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4Cdt2- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4Cdt2-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG.

MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex

MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.

Degradation of p12 subunit by CRL4Cdt2 E3 ligase inhibits fork progression after DNA damage.

Background: The mechanism for inhibiting fork progression after DNA damage still remains. Results: CRL4Cdt2 promotes the degradation of the p12. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis and decreased inhibition of fork progression. Conclusion: p12 degradation by CRL4Cdt2 is critical for inhibiting fork progression. Significance: p12 degradation is one mechanism by which DNA damage in S-phase cells inhibits fork progression. After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1β-TrCP and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4Cdt2 is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4Cdt2 and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.